We have made significant progress in cell- and biomaterial-based skeletal tissue engineering in the last year. (1) Development and application of electrospun nanofibrous scaffold for tissue engineering: - improved cell seeding, application of growth factors, and the use of dynamic bioreactor for efficient cartilage formation - formation of aligned nanofibrous scaffold for mesenchymal stem cell-based engineering of meniscus-like tissue construct - successful repair of osteochondral defect using MSC-nanofibrous composite as well as autologous chondrocyte-loaded biomaterial - amalgam of nanofibrous scaffold and hyaluronan hydrogel for the engineering of biphasic, intervertebral disc-like constructs - adipose tissue engineering using MSCs and nanofibrous scaffold (2) Tissue engineering of tendon/ligament using mesenchymal stem cell-loaded collagen gel under static tension (3) Characterization of physical loading and growth factor effects on chondrocyte and MSC-based cartilage constructs (4) Application of biomaterials for controlled release and delivery of bioactive factors and genetic materials to promote cell differentiation and tissue formation (5) Analysis of Physical Influences on Skeletal Biology. Skeletal tissues are uniquely adapted to responding to mechanical influences. We are currently designing mechanoactive bioreactor systems to apply both dynamic and static mechanical stimulation based on hydrostatic and compressive loading, and using mesenchymal stem cell-based tissue constructs to analyze the cellular and molecular basis of the biological responses. By varying the nature of the biomaterial scaffold, the mixture of cell types, and the growth factor treatment, we aim to decipher the crosstalk among various signal transduction pathways. More importantly, we are examining the interaction between different cell types, e.g., chondrocytes and endothelial cells, under mechanoactive environment. We are also applying a mechanoactive bioreactor system for tendon/ligament tissue engineering. The novel technologies developed in this project have significant application potential for the repair and regeneration of injured and diseased skeletal tissues, in diseases such as osteoarthritis, degenerative disc diseases, tendon rupture, and meniscal tear. In the case of osteoarthritis, we have made substantial progress demonstrating the utility of mesenchymal stem cell-seeded nanofibrous construct in repair an a critical size osteochondral defect in a large animal (pig) model. We are planning a study on the repair of cartilage defects produced by supraphysiological impact, which simulates traumatic osteoarthritis, using similar cell-biomaterial constructs. Similarly, we are excited about the potential utility of the intervertebral disc construct produced using the nanofiber-hydrogel amalgam approach, and will plan a small animal study to evaluate its functionality to repair lacerated intervertebral disc. Overall, we believe that Information gathered from this project should lead to a rational basis to functional skeletal tissue engineering and its application to skeletal diseases of significant burden, including osteoarthritis, degenerative disc diseases, and other soft tissue injuries.